Methods for treating cryptosporidiosis using triazolopyridazines

ABSTRACT

Methods for treating or prophylaxis of a Cryptosporidium infection using compositions comprising a structure disclosed herein. Also provided are pharmaceutical compositions and kits for alleviating the symptoms of, for treating, or for preventing the occurrence of Cryptosporidium infection. The kits comprise one or more compounds having a structure disclosed herein, such as in an oral composition, and instructions for use, storage, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationNo. 62/140,762, filed Mar. 31, 2015, the disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure generally relates to treatment ofcryptosporidiosis. More particularly, the present disclosure relates totriazolopyridazine compounds for treating parasites (e.g.,cryptosporidiosis).

BACKGROUND OF THE DISCLOSURE

The apicomplexan parasites Cryptosporidium parvum and Cryptosporidiumhominis are major etiologic agents of cryptosporidiosis in humans.Infection is typically self-limited in immunocompetent adults, but itcan lead to chronic and fulminant disease in immunocompromised patients,as well as malnutrition and stunting in children. Nitazoxanide is thecurrent standard of care for cryptosporidiosis, but the drug onlyexhibits partial efficacy in children and is no more effective thanplacebo in AIDS patients. Unfortunately, the development of noveltherapeutics for cryptosporidiosis has proven to be extremely difficultas a result of the financial obstacles that plague drug discovery fordiseases that disproportionately affect the developing world, as well astechnical limitations associated with the laboratory study ofCryptosporidium parasites.

While cryptosporidiosis is a significant cause of self-limited diarrheain immunocompetent individuals who may be exposed to parasites throughcontaminated municipal and recreational water supplies or throughoccupational exposures, the burden of cryptosporidiosis is even moresubstantial in immunocompromised and pediatric populations.Immunodeficient individuals, including patients maintained onimmunosuppressive regimens following organ transplantation and AIDSpatients, in particular, risk developing chronic, fulminant, andsometimes fatal disease (especially when CD-4⁺ T-cell counts drop below50 cells/mm³). Diarrhea is also a leading cause of death in childrenunder 5 years of age, and the recent Global Enteric Multicenter Study(GEMS) identified Cryptosporidium as a major cause of life-threateningdiarrhea during the first two years of life. Moreover, cryptosporidiosishas been associated with malnutrition and persistent deficits indevelopment in this population.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to methods for treating or prophylaxis ofa cryptosporidium infection. For example, the method comprisesadministering to an individual diagnosed with or suspected of having acryptosporidium infection or at risk of having cryptosporidiuminfection, a composition comprising a therapeutically effective or aprophylactically effective amount of a compound disclosed herein. Thepresent disclosure also provides pharmaceutical compositions foralleviating the symptoms of, for treating, or for preventing theoccurrence of cryptosporidium infection. In one embodiment, thepharmaceutical compositions are suitable for oral administration. Thepresent disclosure also provides kits for alleviating the symptoms of,for treating, or for preventing the occurrence of cryptosporidiuminfection. The kits comprise one or more compounds of the presentdisclosure (such as in an oral composition) and instructions for use,storage and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structures and in vitro EC₅₀ values for inhibition of C.parvum development in HCT-8 cells for examples of compounds of thepresent disclosure.

FIG. 2 shows the anti-parasitic effect of MMV665917 on C. parvum levelsin mice over a 7 day period. Mice were infected with C. parvum one weekprior to experiment and then administered doses (30 mg/kg) of eitherMMV665917, MMV675977, MMV672987 or DMSO twice daily by oral gavage ondays 1-4. The number of oocysts/mg in collected feces were quantified byqPCR. For each set of data for a day, the bars from left to right are:DMSO, MMV665917, MMV675977, and MMV672987.

FIG. 3 shows an example of a representative dose response curve forMMV665917. The graph shows the relationship between an increasingconcentration of MMV665917 and the corresponding inhibition of C. parvumin vitro.

FIG. 4 provides data of the pertinent physicochemical, pharmacokinetic,and oral bioavailability data for MMV665917. Additionally, hERGinhibition studies with MMV665917 are also included.

FIG. 5 shows data from a repeat of the experiment shown in FIG. 2,except that two different dosing regimens of MMV665917 were used. Afterinfection with C. parvum one week prior to compound dosing, mice weretreated by oral gavage with either MMV665917 at 30 mg/kg twice daily or60 mg/kg twice daily for seven days. Mice treated with DMSO orParomomycin 1000 mg/kg twice daily served as negative and positivecontrols. As before, mice treated with paromomycin relapsed quicklyafter treatment cessation. In this experiment, mice treated with 60mg/kg twice daily MMV665917 were cured, but, in contrast to theexperiment in FIG. 2, 30 mg/kg twice daily MMV665917 was not effective.

FIG. 6 shows a schematic for the experimental strategy to determine ifMMV665917 is static or cidal for C. parvum, and the concentration ofcompound required to maximize the rate of parasite elimination. HCT-8cell monolayers are infected and parasites are allowed to replicate for24 hours before addition of experimental drug compounds. Parasitenumbers are then measured at different time points following compoundaddition by epifluorescence microscopy, using the same method as used inFIG. 1. The cartoon shows the expected results for cidal or staticcompounds, with static compounds simply preventing further expansion ofthe parasite numbers after compound addition and cidal compoundsexpected to result in parasite elimination over time.

FIG. 7 shows the results of experiments to determine if MMV665917 or thecurrent standard-of-care drug, nitazoxanide, are static or cidal againstC. parvum, and the concentration of compound expressed as a multiple ofthe in vitro EC90 that is required to maximize the rate of parasiteelimination. (A) Results for nitazoxanide, which appears to be static,since C. parvum persists indefinitely in the presence of concentrationsup to 12× the EC90. (B) Results for MMV665917, which appears to be cidalfor C. parvum at concentrations equal to or exceeding the EC90. Themaximal rate of parasite elimination is achieved at compounds atconcentrations at or exceeding 3×EC90. Furthermore, since additionalincreases in compound concentration have no effect on the rate ofparasite elimination, it can be inferred that the mode-of-action ofMMV665917 is time-dependent, rather than concentration dependent.

FIG. 8 shows the concentration of MMV665917 in mouse, rat and dairy calfplasma following administration of a single dose. Compounds were dosedas indicated either orally by oral gavage or intravenously, and serumsamples were collected at the indicated time points. The MMV665917plasma concentration was measured by LC-MS/MS. The dashed line shows thepredicted plasma level out to 24 hours for mice, which was determinedusing the calculated elimination half-life.

FIG. 9 shows the actual and simulated mouse and calf plasmaconcentrations for MMV665917, correlated to the EC90 and 3×EC90concentrations. The data relate to those presented in FIGS. 5 and 7.Mouse serum concentrations measured from a single 55 mg/kg oral dose ofMMV665917 (see above figure) were used to simulate the mouse serumconcentrations following BID dosing of either 30 mg/kg or 60 mg/kg. Themeasured calf plasma concentration following a single 22 mg/kg oral doseis also shown. Note that a minimum concentration of EC90 was requiredfor parasite elimination in vitro, and the rate of parasite eliminationin vitro was maximized at 3× the EC90 concentration. Following 30 mg/kgBID dosing in mice, this concentration is not attained and MMV665917 wasvariably efficacious (i.e. effective in FIG. 2 and ineffective in therepeat experiment in FIG. 5). A 60 mg/kg BID dose, on the other hand,results in a sustained plasma concentration greater than the EC90, andis curative in the NOD SCID gamma mouse model (see FIG. 5).

DETAILED DESCRIPTION OF THE DISCLOSURE

In an aspect, the present disclosure provides a method for treating acryptosporidium infection or a method for prophylaxis comprisingadministering to an individual a therapeutically effective amount of acompound having a [1,2,4]triazolo[4,3-b]pyridazine (henceforth referredto as “triazolopyridazine”) scaffold.

As used herein, the term “alkyl group,” unless otherwise stated, refersto branched or unbranched hydrocarbons. Examples of such alkyl groupsinclude methyl groups, ethyl groups, propyl groups, butyl groups,iso-propyl groups, sec-butyl, tert-butyl groups, and the like. Forexample, the alkyl group is a C₁ to C₉ alkyl group, including allinteger numbers of carbons and ranges of numbers of carbonstherebetween. Alkyl groups can be substituted with various otherfunctional groups. The alkyl groups may be substituted with groups suchas, for example, amines (acyclic and cyclic), alcohol groups, ethergroups, and halogen atoms.

As used herein, unless otherwise indicated, alkoxy means

where R^(a) is a linear, branched or cyclic C₁-C₆ alkyl group, includingall integer numbers of carbons and ranges of numbers of carbonstherebetween. For example, suitable alkoxy groups include methoxy,ethoxy, propoxy, iso-propoxy, butoxy, sec-butoxy, tert-butoxy, andhexoxy groups. Additionally, alkyl substituents can be substituted withvarious other functional groups, e.g. functional groups disclosedherein.

As used herein, unless otherwise indicated, amino means

where each R^(b) is selected independently from the group consisting ofhydrogen atom, substituted or unsubstituted C₁-C₁₀ alkyl, including allinteger numbers of carbons and ranges of numbers of carbonstherebetween, substituted or unsubstituted phenyl, substituted orunsubstituted heteroaryl, substituted carbonyl, substituted sulfonyl,haloalkyl, and substituted or unsubstituted benzyl groups.

As used herein, unless otherwise indicated, benzyl group means

where R^(c) is a substituent on the phenyl ring and n is from 0 to 5.The substituents can be the same or different. For example, thesubstituents on the benzyl group include substituted or unsubstitutedalkyl, —NH₂, phenyl, haloalkyl (e.g., —CF₃), halo (e.g., —F, —Cl, —Br,—I), alkoxy (e.g., —OMe), and —OH groups.

As used herein, the term “cycloalkyl” refers to a cyclic hydrocarbongroup, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentylgroups. Cycloalkyl groups can be saturated or partially unsaturated ringsystems optionally substituted with, for example, one to three groups.Each group is independently selected from the group consisting of alkyl,—NH₂, oxo (═O), phenyl, haloalkyl (e.g., —CF₃), halo (e.g., —F, —Cl,—Br, —I), alkoxy, and —OH groups. Additionally, alkyl substituents maybe substituted with various other functional groups.

As used herein, unless otherwise indicated, halogen means fluorine,chlorine, bromine, and iodine, and halo means fluoro, chloro, bromo, andiodo.

As used herein, the term “heteroaryl” refers to a monocyclic or bicyclicring system comprising one or two aromatic rings and containing at leastone nitrogen or oxygen atom in an aromatic ring. Unless otherwiseindicated, a heteroaryl group can be unsubstituted or substituted withone or more, and in particular one or two, substituents selected fromthe group consisting of, for example, haloalkyl (e.g., —CF₃), halo(e.g., —F, —Cl, —Br, —I), alkyl, alkoxy, amino, —CO₂H, —CO₂alkyl, aryl,and heteroaryl groups. Examples of heteroaryl groups include,benzofuranyl, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl,isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl,imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, andthiadiazolyl groups.

As used herein, unless otherwise indicated, —OPh (phenoxy) means

where each Y is independently selected from the group consisting of F,Cl, Br, and I and m can be 0, 1 or 2.

As used herein, unless otherwise indicated, phenyl group means

where each R^(d) is an independent substituent on the phenyl group and nis from 0 to 5. The substituents at different occurrences can be thesame or different. For example, the substituents on the phenyl groupinclude substituted or unsubstituted C₁-C₆ alkyl, including all integernumbers of carbons and ranges of numbers of carbons therebetween,substituted or unsubstituted amino, haloalkyl (e.g., —CF₃), halo (e.g.,—F, —Cl, —Br, —I), substituted or unsubstituted alkoxy (e.g., —OMe), andsulfonyl group. In certain instances, two adjacent R groups can beconnected through to form a dioxolyl group.

As used herein, unless otherwise indicated, sulfonyl group means

where R^(e) can be a linear, branched or cyclic alkyl group, or aminogroup of the structure

where each R^(f) is independently selected from a hydrogen atom, linear,branched and cyclic alkyl group. They may also combine to form

In an aspect, the present disclosure provides a methods for treating anindividual diagnosed with or suspected of having a cryptosporidiuminfection. Compounds of the present disclosure can be used in themethods.

In an embodiment, a method for treating an individual diagnosed with orsuspected of having a cryptosporidium infection comprises administeringto the individual a therapeutically effective amount of a compoundhaving a composition comprising the following structure:

where R¹ is selected from the group consisting of hydrogen atom,substituted or unsubstituted C₁-C₈ alkyl group, sulfonyl group havingthe structure: —S(O)₂R, and carbonyl containing group having thestructure: —C(O)R, —C(O)₂R, or —C(O)N(R)₂, where R is independentlyselected from the group consisting of hydrogen atom, substituted orunsubstituted C₁-C₉ alkyl group, including all integer numbers ofcarbons and ranges of numbers of carbons therebetween, substituted orunsubstituted heteroaryl group, substituted or unsubstituted alkoxygroup, substituted or unsubstituted amino group, substituted orunsubstituted phenyl group, substituted or unsubstituted benzyl group,substituted or unsubstituted heteroaryl group, and substituted orunsubstituted —CH₂-heteroaryl group comprising 1 to 4 O and/or N atoms;R² is a hydrogen atom, or it may also combine with R¹ to form

R³ is a hydrogen atom, or a substituted or unsubstituted C₁ to C₉ alkylgroup, including all integer numbers of carbons and ranges of numbers ofcarbons therebetween R⁴ is selected from the group consisting ofhydrogen atom, substituted or unsubstituted C₁ to C₉ alkyl group,including all integer numbers of carbons and ranges of numbers ofcarbons therebetween, C₃-C₆ cycloalkyl group, substituted orunsubstituted C₁-C₆ haloalkyl group, including trifluoromethyl group,and phenyl derivative having the structure:

where Y is independently selected from the group consisting of F, Cl,Br, and I, and n can be 0, 1, 2, 3, 4, or 5. X is CH₂ or C═O; and a is 1or 2.

In an embodiment, the compound has the following structure:

where R⁵ is selected from the group consisting of substituted orunsubstituted alkyl group, substituted or unsubstituted C₃ to C₆cycloalkyl group, substituted or unsubstituted benzyl group, substitutedor unsubstituted amino group, substituted or unsubstituted phenyl group,substituted or unsubstituted alkoxy group, substituted or unsubstitutedalkylheterocyclic group, substituted or unsubstituted phenoxy group, andsubstituted or unsubstituted aniline group. In various embodiments R⁵ isselected from the group consisting of:

In an embodiment, the compound has the following structure:

wherein Y is a bond, S(O)₂, or CH₂; R⁶ is selected from the groupconsisting of substituted or unsubstituted C₁-C₈ alkyl group, includingall integer numbers of carbons and ranges of numbers of carbonstherebetween, substituted or unsubstituted cycloalkyl group, substitutedor unsubstituted benzyl group, substituted or unsubstituted phenylgroup, and substituted or unsubstituted heteroaryl group; X is CH₂ orC═O; and a is 1 or 2.

In an embodiment, the compound has the following structure:

where R⁷ is a substituted or unsubstituted C₁-C₈ alkyl group.

In an embodiment, the compound has the following structure:

In an embodiment, the compound has the following structure:

In an embodiment, the compound has the following structure:

In an embodiment, the compound has the following structure:

In an embodiment, the compound has the following structure:

where Z is independently selected from the group consisting of hydrogenand fluorine atom.

The compounds referred to in this disclosure as MMV665917, MMV675977,and MMV672987 have the following structures:

In an embodiment, the disclosure provides a method for treating anindividual diagnosed with or suspected of having a cryptosporidiuminfection comprising administering to the individual a therapeuticallyeffective amount of one or more of the following compounds:

The compounds exhibit wide variability in pharmacokinetic andphysicochemical properties while still retaining desirable biologicalactivity as described herein (see, for example, FIG. 4). For examplesolubility, e.g., log P, is variable while still retaining desirablebiological activity as described herein.

The compounds may be used to treat a cryptosporidium infection caused byany species of Cryptosporidium. Examples of Cryptosporidium speciesinclude, Cryptosporidium parvum or Cryptosporidium hominis.

The individual to be treated by the method of the disclosure may behuman or non-human. Non-human animals include ungulates such as bovines.

In an aspect the present disclosure provides a composition foradministration to an individual. For example a composition comprises apH sensitive polymer.

In an embodiment the composition comprises a compound of the presentdisclosure encapsulated in a pH sensitive polymer suitable for releaseof a compound of the disclosure in the small intestines, distal smallintestine, or colon.

In an embodiment, the pH sensitive polymer suitable for such a releasecan be a synthetic anionic polymer based on a monomer such as acrylicacid, methacrylic acid, propionic acid,2-acrylmido-2-methylpropylsulfonic acid, 2-methacryloxyethylsulfonicacid, 3-methacryloxy-2-hydroxypropylsulfonic acid, ethylenesulfonicacid, styrenesulfonic acid, sulfoxyethyl methacrylate, or a combinationthereof. In another embodiment, the anionic polymer can be a naturalanionic polymer such as hyaluronic acid, alginic acid, carboxymethylcellulose, carboxymethyl dextran, poly(aspartic acid), or heparin.Several commercial pH sensitive polymers are available. For example,Eudragit L, Eudragit S from Röhm Pharma GmBH (based on methacrylic acidand methyl methacrylate) or CMEC from Freund Sangyo Co., Ltd; CAP fromWako Pure Chemicals Ltd.; or HP-50 and ASM from Shin-Etsu Chemical Co.,Ltd. (derived from cellulose) can be used.

Without intending to be bound by any particular theory it is consideredthat compounds with adequate solubility to access the parasites withinintestinal epithelial cells are desired. In an embodiment, the compoundsare largely retained at the site of infection within the gut. Consistentwith the ability of achieving this goal, the SAR studies conducteddemonstrate that formulations of each of these chemical scaffolds withwidely divergent solubility (and, therefore, systemic absorption) haveretained potency against Cryptosporidium within intestinal epithelialcells. In essence, compounds for treatment of cryptosporidiosis shouldbreak several of Lipinski's rules for selection of drug leads. Thus,formulations of each scaffold that are optimized for treatment ofcryptosporidiosis will be distinct from formulations appropriate fortreatment of a systemic infection such as malaria. Drug exposure (boththe ability to penetrate the cell and parasitophorous vacuole, andretention at the site of infection rather than oral absorption) will beequivalent. In an embodiment, the compounds are not releasedsystemically but are rather retained in the gastrointestinal tract. Inan embodiment, the compounds of the method are taken up by the cells ofthe lumen of the gastrointestinal tract. In another embodiment, systemicexposure may be necessary in certain cases. For example, in severelyimmunocompromised people such as those with AIDS, infection can involvethe biliary tree and, rarely, even the lungs. In these circumstances, adrug or formulation that favors systemic absorption, and/orenterohepatic recirculation may be desirable.

In an embodiment, a formulation comprising a compound of the disclosureis formulated in a manner such that an extended release in the smallintestines, distal small intestine, or colon is achieved. For example,pH sensitive polymers can be used as described herein.

In an aspect, the present disclosure provides a composition comprisingat least one compound of the disclosure. Compositions comprising atleast one compound of the disclosure include, for example,pharmaceutical preparations.

The present disclosure includes all possible stereoisomers and geometricisomers of a compound of the present disclosure. The present disclosureincludes both racemic compounds and optically active isomers. When acompound of the present disclosure is desired as a single enantiomer, itcan be obtained either by resolution of the final product or bystereospecific synthesis from either isomerically pure starting materialor use of a chiral auxiliary reagent, for example, see Z. Ma et al.,Tetrahedron: Asymmetry, 8(6), pages 883-888 (1997). Resolution of thefinal product, an intermediate, or a starting material can be achievedby any suitable method known in the art. Additionally, in situationswhere tautomers of a compound of the present disclosure are possible,the present disclosure is intended to include all tautomeric forms ofthe compounds.

Prodrugs of a compound of the present disclosure also can be used as thecompound in a method of the present disclosure. It is well establishedthat a prodrug approach, wherein a compound is derivatized into a formsuitable for formulation and/or administration, then released as a drugin vivo, has been successfully employed to transiently (e.g.,bioreversibly) alter the physicochemical properties of the compound(see, H. Bundgaard, Ed., “Design of Prodrugs,” Elsevier, Amsterdam,(1985); R. B. Silverman, “The Organic Chemistry of Drug Design and DrugAction,” Academic Press, San Diego, chapter 8, (1992); K. M. Hillgren etal., Med. Res. Rev., 15, 83 (1995)).

Compounds of the present disclosure can contain one or more functionalgroups. The functional groups, if desired or necessary, can be modifiedto provide a prodrug. Suitable prodrugs include, for example, acidderivatives, such as amides and esters. It also is appreciated by thoseskilled in the art that N-oxides can be used as a prodrug.

Compounds of the disclosure can exist as salts. Pharmaceuticallyacceptable salts of the compounds of the disclosure generally arepreferred in the methods of the disclosure. As used herein, the term“pharmaceutically acceptable salts” refers to salts or zwitterionicforms of a compound of the present disclosure. Salts of compounds havingthe structure (I) to (XII) can be prepared during the final isolationand purification of the compounds or separately by reacting the compoundwith an acid having a suitable cation. The pharmaceutically acceptablesalts of a compound of the present disclosure are acid addition saltsformed with pharmaceutically acceptable acids. Examples of acids whichcan be employed to form pharmaceutically acceptable salts includeinorganic acids such as nitric, boric, hydrochloric, hydrobromic,sulfuric, and phosphoric, and organic acids such as oxalic, maleic,succinic, and citric. Nonlimiting examples of salts of compounds of thedisclosure include, the hydrochloride, hydrobromide, hydroiodide,sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogenphosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate,butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate,hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate,maleate, ascorbate, isethionate, salicylate, methanesulfonate,mesitylenesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate,trifluoroacetate, phosphate, glutamate, bicarbonate,paratoluenesulfonate, undecanoate, lactate, citrate, tartrate,gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, andp-toluenesulfonate salts. In addition, available amino groups present inthe compounds of the disclosure can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides. Inlight of the foregoing, any reference to compounds of the presentdisclosure appearing herein is intended to include a compound of thepresent disclosure as well as pharmaceutically acceptable salts,hydrates, or prodrugs thereof.

Compositions comprising a compound of the disclosure and apharmaceutical agent can be prepared at a patient's bedside, or by apharmaceutical manufacture. In the latter case, the compositions can beprovided in any suitable container, such as a sealed sterile vial orampoule, and may be further packaged to include instruction documentsfor use by a pharmacist, physician or other health care provider. Thecompositions can be provided as a liquid, or as a lyophilized or powderform that can be reconstituted if necessary when ready for use. Inparticular, the compositions can be provided in combination with anysuitable delivery form or vehicle, examples of which include, forexample, liquids, caplets, capsules, tablets, inhalants or aerosol, etc.The delivery devices may comprise components that facilitate release ofthe pharmaceutical agents over certain time periods and/or intervals,and can include compositions that enhance delivery of thepharmaceuticals, such as nanoparticle, microsphere or liposomeformulations, a variety of which are known in the art and arecommercially available. Further, each composition described herein cancomprise one or more pharmaceutical agents. The compositions describedherein can include one or more standard pharmaceutically acceptablecarriers. Some examples of pharmaceutically acceptable carriers can befound in: Remington: The Science and Practice of Pharmacy (2005) 21stEdition, Philadelphia, Pa. Lippincott Williams & Wilkins.

Examples of pharmaceutically-acceptable carrier includepharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The dose of the composition comprising a compound of the disclosure anda pharmaceutical agent generally depends upon the needs of theindividual to whom the composition of the disclosure is to beadministered. These factors include, for example, the weight, age, sex,medical history, and nature and stage of the disease for which atherapeutic or prophylactic effect is desired. The compositions can beused in conjunction with any other conventional treatment modalitydesigned to improve the disorder for which a desired therapeutic orprophylactic effect is intended, non-limiting examples of which includesurgical interventions and radiation therapies. The compositions can beadministered once, or over a series of administrations at variousintervals determined using ordinary skill in the art, and given thebenefit of the present disclosure.

Compositions of the disclosure can comprise more than one pharmaceuticalagent. For example, a first composition comprising a compound of thedisclosure and a first pharmaceutical agent can be separately preparedfrom a composition which comprises the same compound of the disclosureand a second pharmaceutical agent, and such preparations can be mixed toprovide a two-pronged (or more) approach to achieving the desiredprophylaxis or therapy in an individual. Further, compositions of thedisclosure can be prepared using mixed preparations of any of thecompounds disclosed herein.

It is envisioned, therefore, that a compound of the present disclosureis useful in the treatment of a cryptosporidium infection. Thus, thepresent disclosure concerns the use of a compound of the presentdisclosure, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing either a compound of the presentdisclosure, or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament for the treatment of such conditions anddiseases.

Cryptosporidium infection is defined as detection of Cryptosporidium inthe feces, by any standard means such as microscopic parasite exam,antigen detection, and polymerase chain reaction. In some instances,such individuals may be suffering from diarrhea, but may be asymptomaticwhere there are indications to treat for prevention of long-termsequelae (such as malnutrition and growth stunting) and for preventionof spreading infection to others. In areas where Cryptosporidiuminfection are highly endemic, treatment for cryptosporidiosis may beprovided (alone or in combination with treatment for other entericinfections) for suspected infection without microbiologic confirmation.

The compounds of the present disclosure can be therapeuticallyadministered as the neat chemical, but it is preferred to administer acompound of the present disclosure as a pharmaceutical composition orformulation. Thus, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the present disclosure togetherwith a pharmaceutically acceptable diluent or carrier therefore. Alsoprovided is a process of preparing a pharmaceutical compositioncomprising admixing a compound of the present disclosure with apharmaceutically acceptable diluent or carrier therefore.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

In one embodiment, the pharmaceutically-acceptable formulation is suchthat it provides sustained delivery of a compound of the presentdisclosure to an individual for at least 12 hours, 24 hours, 36 hours,48 hours, one week, two weeks, three weeks, or four weeks after thepharmaceutically-acceptable formulation is administered to theindividual.

In certain embodiments, these pharmaceutical compositions are suitablefor oral administration to an individual. In other embodiments, asdescribed in detail below, the pharmaceutical compositions of thepresent disclosure may be specially formulated for administration insolid or liquid form, including those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, boluses, powders, granules, and pastes.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon theindividual being treated, the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof a compound of the present disclosure which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, more preferablyfrom about 10 percent to about 30 percent.

Methods of preparing these compositions include the step of bringinginto association a compound of the present disclosure with the carrierand, optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a compound of the present disclosure with liquid carriers,or finely divided solid carriers, or both, and then, if necessary,shaping the product.

Compositions of the disclosure suitable for oral administration may bein the form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent disclosure as an active ingredient. A compound of the presentdisclosure may also be administered as a bolus, electuary or paste.

In solid dosage forms of the disclosure for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present disclosure, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of a compound of the presentdisclosure include pharmaceutically-acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to a compound of the disclosure, thecomposition may contain suspending agents as, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

When a compound of the present disclosure are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically-acceptable carrier.

In certain embodiments, the methods of the disclosure includeadministering to an individual a therapeutically effective amount of acompound of the present disclosure in combination with anotherpharmaceutically active ingredient. Pharmaceutically active ingredientsthat may be used can be found in Harrison's Principles of InternalMedicine, Thirteenth Edition, Eds. T. R. Harrison et al. McGraw-HillN.Y., N.Y.; and the Physicians Desk Reference 50th Edition 1997, OradellN.J., Medical Economics Co., the complete contents of which areexpressly incorporated herein by reference. A compound of the presentdisclosure and the pharmaceutically active ingredient may beadministered to the individual in the same pharmaceutical composition orin different pharmaceutical compositions (at the same time or atdifferent times).

Methods delineated herein include those wherein the individual isidentified as in need of a particular stated treatment. Identifying anindividual in need of such treatment can be in the judgment of anindividual or a health care professional and can be subjective (e.g.opinion) or objective (e.g. measurable by a test or diagnostic method).In other methods, the individual is prescreened or identified as in needof such treatment by assessment for a relevant marker or indicator ofsuitability for such treatment.

The compounds and compositions disclosed in the present disclosure canalso be used for prophylaxis in an individual who is at risk of beingexposed to cryptosporidium. A prophylactic use may be useful, forexample, in an individual who is about to undertake a journey to aregion where an outbreak of cryptosporidium has been reported or isknown to occur. For the prophylactic use, one or more doses of acomposition comprising the compounds of the present disclosure may beadministered. Dosing for prophylactic treatment may vary compared todosing of known cryptosporidium infections. In one embodiment treatmentmay be given 1-3 times a day for 1 to 10 days or longer. In oneembodiment, treatment is given for up to three times (such as 1 to 3times) daily for up to 10 days (such as 1-10 days) in immunocompetenthosts. For immmuosuppressed hosts such as those with AIDS, long-termsuppressive treatment may be warranted.

The identification of those patients who are in need of prophylactictreatment for a Cryptosporidium infection can readily identify suchcandidate patients, by the use of, for example, clinical tests, physicalexamination and medical/family history. The individual may have aCryptosporidium infection, may be at risk of developing aCryptosporidium infection, or may need prophylactic treatment prior toanticipated or unanticipated exposure to a condition(s) capable ofincreasing susceptibility to a Cryptosporidium infection. In anembodiment, those in need of prophylactic treatment for aCryptosporidium infection can take a therapeutically effective amount ofa compound of the present disclosure from 1 to 30 days prior to ananticipated or unanticipated exposure to a condition(s) capable ofincreasing susceptibility to a Cryptosporidium infection. In anotherembodiment, those in need of prophylactic treatment for aCryptosporidium infection can take a therapeutically effective amount ofa compound of the present disclosure from 1 to 24 hours prior to ananticipated or unanticipated exposure to a condition(s) capable ofincreasing susceptibility to a Cryptosporidium infection. In anembodiment, an individual can take a therapeutically effective amount ofa compound of the present disclosure shortly after exposure toCryptosporidium infection.

In another aspect, the disclosure provides a packaged compositionincluding a therapeutically effective amount of a compound of thepresent disclosure and a pharmaceutically acceptable carrier or diluent.The composition may be formulated for treating an individual sufferingfrom or susceptible to a Cryptosporidium infection, and packaged withinstructions to treat an individual suffering from or susceptible to aCryptosporidium infection.

In one aspect, the disclosure provides a kit for treating aCryptosporidium infection in an individual is provided and includes acompound of the present disclosure, pharmaceutically acceptable esters,salts, and prodrugs thereof, and instructions for use. In certainembodiments, the disclosure provides: a kit for treating aCryptosporidium infection, in an individual, the kit comprising acompound of the present disclosure. The kit may also includeinstructions for administration of the compound or composition. Theinstructions may include details on one or more of the following:dosage, frequency, number of administrations to be carried out (such asnumber of tablets to be consumed), whether the composition needs to betaken with food, water etc., storage of the composition, and the like.

For veterinary use, a compound of the present disclosure, or apharmaceutically acceptable salt or prodrug, is administered as asuitably acceptable formulation in accordance with normal veterinarypractice. The veterinarian can readily determine the dosing regimen androute of administration that is most appropriate for a particularanimal. Animals treatable by the present compounds and methods include,but are not limited to, bovines or ungulates.

When administered in combination with other therapeutics, a presentcompound may be administered at relatively lower dosages. In addition,the use of targeting agents may allow the necessary dosage to berelatively low. Certain compounds may be administered at relatively highdosages due to factors including, but not limited to, low toxicity andhigh clearance.

For human use, a compound of the present disclosure can be administeredalone, but generally is administered in admixture with a pharmaceuticalcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice. Pharmaceutical compositions for use inaccordance with the present disclosure can be formulated in aconventional manner using one or more physiologically acceptable carriercomprising excipients and auxiliaries that facilitate processing of acompound of the present disclosure into pharmaceutical preparations.

In an aspect, a compound as described herein exhibits activity againstCryptosporidium. In certain embodiments, the Cryptosporidium infectionis from the Cryptosporidium parvum, Cryptosporidium hominis,Cryptosporidium andersoni, or a combination thereof. In variousembodiments, the compounds of the disclosure are those which display invitro IC₅₀ values upwards of 4.54 μM against Cryptosporidium parvum.

As appreciated by persons skilled in the art, additional active orancillary agents can be used in the methods described herein. Referenceherein to treatment also extends to prophylaxis, as well as to treatmentof established diseases or symptoms.

The present disclosure can be applied to cell populations ex vivo. Forexample, the present compounds can be used ex vivo to determine theoptimal schedule and/or dosing of administration of the present compoundfor a given indication, cell type, patient, and other parameter.Information gleaned from such use can be used for experimental purposesor in the clinic to set protocol for in vivo treatment. Other ex vivouses for which the disclosure is suited are apparent to those skilled inthe art.

The steps of the method described in the various embodiments andexamples disclosed herein are sufficient to treat an individualdiagnosed with or suspected of having a cryptosporidium infection or amethod for prophylaxis in an individual diagnosed with or suspected ofhaving a cryptosporidium infection. Thus, in an embodiment, the methodconsists essentially of a combination of the steps of the methoddisclosed herein. In another embodiment, the method consists of suchsteps.

In the following Statements, various examples of the methods,compositions, and kits of the present disclosure are described:

-   1. In an example, a method for treating an individual diagnosed with    or suspected of having a cryptosporidium infection or prophylaxis in    an individual who is at risk of having a cryptosporidium infection    comprises administering (e.g., by any method disclosed herein) to    the individual (e.g., an individual disclosed herein) a compound or    a composition comprising a therapeutically effective or a    prophylactically effective amount of the compound having the    following structure:

where R¹ is selected from the group consisting of hydrogen atom, C₁-C₈alkyl group, sulfonyl group having the structure: —S(O)₂R, and carbonylcontaining group having the structure: —C(O)R, —C(O)₂R, or —C(O)N(R)₂,where R is independently at each occurrence selected from the groupconsisting of hydrogen atom, C₁-C₉ alkyl group, heteroaryl group, alkoxygroup, amino group, phenyl group, benzyl group, heteroaryl group, and—CH₂-heteroaryl group comprising 1 to 4 O and/or N atoms;

-   R² is a hydrogen atom, or it may also combine with R¹ to form

-   R³ is a hydrogen atom or a C₁-C₉ alkyl group; R⁴ is selected from    the group consisting of hydrogen atom, C₁-C₉ alkyl group, C₃-C₆    cycloalkyl group, haloalkyl group, and phenyl derivative having the    structure:

where Y is independently selected from the group consisting of F, Cl,Br, and I, and n can be 0, 1, 2, 3, 4, or 5; X is —CH₂ or C═O; and

-   a is 1 or 2.-   2. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

where R⁵ is selected from the group consisting of alkyl group, C₃ to C₆cycloalkyl group, benzyl group, amino group, phenyl group, alkoxy group,alkylheterocyclic group, phenoxy group, and aniline group.

-   3. In another example, a method is the method of Statement 2 in    which R⁵ is selected from the group consisting of the R⁵ groups    disclosed herein.-   4. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

where Y is a bond, —S(O)₂, or —CH₂; R⁶ is selected from the groupconsisting of C₁-C₈ alkyl group, cycloalkyl group, benzyl group, phenylgroup, and heteroaryl group; X is —CH₂ or C═O; and a is 1 or 2.

-   5. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

where R⁷ is a C₁-C₈ alkyl group.

-   6. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

-   7. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

-   8. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

-   9. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

-   10. In another example, a method is the method of Statement 1 in    which the compound has the following structure:

wherein Z is independently selected from the group consisting ofhydrogen and fluorine atom.

-   11. In another example, a method is the method of Statement 1 in    which the compound is selected from a compound disclosed herein and    combinations thereof.-   12. In another example, a method is the method of any one of the    preceding Statements in which the cryptosporidium infection is    caused by Cryptosporidium parvum, Cryptosporidium hominis, or    Cryptosporidium andersoni.-   13. In another example, a method is the method of any one of the    preceding Statements in which the individual is human or non-human    animal (e.g., bovine, or ungulate).-   14. In an example, a composition for oral administration comprises a    compound disclosed herein (e.g., a compound of any one of the    preceding Statements) encapsulated in a pH sensitive polymer    suitable for release of the compound in the small intestines, distal    small intestine, or colon.-   15. In an example, a kit comprises a compound or a composition    disclosed herein (e.g., a compound or composition of any one of the    preceding Statements) and instructions on administration details to    an individual who has been diagnosed with or who is at risk of    getting cryptosporidium infection wherein said details comprise one    or more of the following: dosage, frequency, and length of time for    administration of the composition.

The following examples are presented to illustrate the presentdisclosure. They are not intended to be limiting in any manner.

Example 1

In this example, a cell-based screening assay was utilized to identifynovel Cryptosporidium parvum growth inhibitors, and an immunocompromisedmouse model for in vivo validation of identified drug screening hits wasidentified.

Materials and Methods:

Cell culture and Cryptosporidium infections: The high throughput screenfor C. parvum growth inhibitors was carried out using human ileocecaladenocarcinoma (HCT-8) cells (ATCC) maintained in RPMI 1640 medium withHEPES, sodium pyruvate (1 mM) and L-glutamate with 10% horse serum(ATCC) supplemented with 120 U/mL penicillin and 120 μg/mL streptomycin.HCT-8 cells were grown to confluence in 384-well, tissueculture-treated, black-walled, clear-bottom microwell plates (BDFalcon). Cells were then inoculated with 5.5×10³/well C. parvum(Bunchgrass Farms, Deary, Id.) oocysts, which had been primed forexcystation with 10 mM HCl and 2 mM sodium taurocholate and thenresuspended in inoculation medium (as described above without horseserum). Infected cells were incubated for three hours at 37° C. at whichpoint an equal volume of growth medium (with experimental compoundsadded in the case of confirmatory assays or without in the case of theHTS; see below) with 20% horse serum was added, and cells were incubatedfor an additional 48 hours (except for minimum effective exposureexperiments, which were incubated for 72 hours).

Experimental Compounds:

The MMV Open Access Malaria Box was diluted to a final concentration of1.67 mM and arrayed into the center 308 wells of V-bottom polypropylene384-well source plates (Whatman) and stored at −80° C. until use. Sourceplates were warmed to 37° C. and briefly centrifuged prior to use. A 384solid pin Multi-Blot replicator tool (V&P scientific) was used totransfer approximately 70 nL of compound to the assay plate for a finalconcentration of approximately 2.3 μM. Controls on each plate includedwells containing DMSO only (vehicle) and nitazoxanide at a stockconcentration that resulted in a final concentration approximately equalto the 90% inhibitory concentration (IC₉₀=6.6 μM).

Hit Definition:

A screening hit was defined as any compound that inhibited the averageC. parvum parasite count from duplicate screening plates by greater than65%, a cutoff that corresponded to reduction by greater than 2 standarddeviations from the mean reduction in parasite counts from the compoundlibrary as a whole.

In Vitro Dose Response Assays:

Compounds that met the hit definition above were purchased fromcommercial sources and used for secondary in vitro and in vivo studies.Purchased compounds were used to generate in vitro dose-response curvesusing varied concentrations for each compound.

Immunofluorescence Assay for C. parvum Detection:

Infected monolayers were washed three times with PBS containing 111 mMD-galactose using an EL406 automated plate washer (Biotek), fixed with4% paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBS, andthen blocked with 4% bovine serum albumin (BSA). Vegetative forms of C.parvum were detected by the addition of biotinylated Vicia villosalectin (VVL; Vector Laboratories) and streptavidin-conjugated AlexaFluor 568 (Invitrogen) diluted in PBS with 1% BSA and 0.1% Tween 20.Nuclei were counterstained with 0.29 mM Hoechst 33258 (Anaspec), andwashed five times with PBS containing 0.1% Tween 20. Plates were thenimaged with a Nikon Eclipse Ti2000 epifluorescent microscope withmotorized stage and Exi blue fluorescent microscopy camera (QImaging).NIS-Elements Advanced Research software (Nikon USA) was used to automateacquisition of a three-by-three 20× field image from the center of eachof the middle 308 wells of the assay plate (corresponding toapproximately 13% of the total surface area of the well). Images wereexported into ImageJ, which was used to execute the batch processfunction to execute previously validated macros to enumerate nuclei andparasites.

Data Handling and Analysis:

ImageJ outputs were imported into Microsoft Excel for data organizationand analysis. Percent inhibition was calculated (equation 1) for eachwell in order to normalize the data and to facilitate the combination ofdata from individual biological replicates. Additional analysis andfigure construction was conducted using GraphPad Prism6 v. 6.00 andVortex

$\begin{matrix}{{\%\mspace{14mu}{inhibition}} = \frac{\begin{matrix}{{\#\mspace{14mu}{parasites}_{{DMSO}\mspace{14mu}{treated}\mspace{14mu}{wells}}} -} \\{\#\mspace{11mu}{parasites}_{{{experimental}\mspace{14mu}{compound}\mspace{14mu}{treated}\mspace{14mu}{wells}}\;}}\end{matrix}}{\#\mspace{11mu}{parasites}_{{DMSO}\mspace{14mu}{treated}\mspace{14mu}{wells}}}} & (1)\end{matrix}$

Dose Response Curves.

Parasite counts were averaged for each infected well treated with adifferent concentration of experimental compound (n=11 wells) and 3uninfected wells treated with the corresponding concentration of drug(to assess for non-specific background staining). The average value forall uninfected wells was subtracted from all parasite counts to adjustfor background signal. Percent inhibition values were averaged for allof the wells in each treatment condition and the data from at least twobiological replicates were combined to generate dose-response curves inGraphPad Prism. IC₅₀ values were calculated using the log[inhibitor]versus response—variable slope equation (equation 2) with the bottom andtop constraints set equal to 0 and 100, respectively.

$\begin{matrix}{Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + 10^{{{LogIC}\; 50} - {X*{Hill}\mspace{11mu}{Slope}}}} \right)}}} & (2)\end{matrix}$

NOD SCID Gamma Immunocompromised Mouse Model of Cryptosporidiosis:

A NOD SCID gamma mouse model was used for in vivo testing of confirmedscreening hits. NOD SCID gamma mice (Jackson Laboratories) developCryptosporidium parvum infection following oral gavage that ischaracterized by persistent infection of the small intestinal and cecalepithelium. The mice remain asymptomatic and gain weight normally.Infection is established by oral gavage of 1×10⁴ C. parvum (Iowa strain)oocysts on day −6. Fecal parasite shedding is quantified by quantitativePCR of genomic DNA, using a standard curve generated by spiking oocystsinto fecal samples from uninfected mice. Fecal oocyst shedding reliablybecomes detectable at day 1 (1 week after infection). Experimentalcompounds are administered by oral gavage of 30 mg/kg in DMSO:1% HPMC(5:95) twice daily for 4 days. Parasite shedding is compared to that forinfected mice treated with drug carrier alone (i.e. DMSO:1% HPMC(5:95)).

Results.

Structures and in vitro EC₅₀ values for inhibition of C. parvumdevelopment in HCT-8 cells for screening hits with confirmed activitycan be seen in FIG. 1. EC₅₀ values of these hits ranged from 0.11 μM to6.4 μM, with MMV665917 demonstrating in vitro potency.

Results for treatment of C. parvum infected NOD SCID gamma mice withDMSO or selected MMV Malaria Box screening hits with confirmed in vitroactivity given by oral gavage of 30 mg/kg twice daily on days 1-4 can beseen in FIG. 2 s. Cryptosporidium parvum infection was established onday −6. Parasite shedding is detectable in all animals on day 1.MMV665917 significantly reduced parasite shedding, which persistedfor >3 days following cessation of treatment. Other compounds and DMSOhad no effect. Data shown are the mean and SE for parasites detected/mgof feces (n=4 animals per treatment group).

A repeat of the experiment presented in FIG. 2 showing efficacy ofMMV665917 in the NOD SCID gamma mouse model is presented in FIG. 5. Onrepeat, 30 mg/kg twice daily was inactive, but 60 mg/kg twice dailyeliminated detection of C. parvum shedding in the feces by real timePCR. Unlike the control compound paromomycin, no relapse was observedover the week following cessation of treatment. For this experiment, NODSCID gamma mice were infected on day −6 by oral gavage of C. parvumoocysts, shedding was detected at day 0 prior to compound dosing,compounds or vehicle were dosed on days 0-6 as indicated, and mice weremaintained for one week following cessation of treatment. Data shown arethe mean and SE for the number of oocysts/mg stool detected by real timePCR of the feces on the indicated day (four mice per experimentalgroup). The variable efficacy of MMV665917 dosed at 30 mg/kg twice dailyis likely due to pharmacokinetic and pharmacodynamic factors detailed inFIGS. 6 through 9. In vitro data indicate that a compound concentrationof greater than or equal to the EC90 concentration is required toachieve cidal anti-parasitic activity (i.e. parasite elimination), andthat parasite elimination is time-dependent and thus requires sustainedcompound concentrations in excess of the EC90 concentration (see FIGS. 6and 7). Based on measurement of serum compound concentrations and levelmodeling (see FIGS. 8 and 9), a twice daily dose of 60 mg/kg achievesconcentrations in excess of the EC90 at all times, and reliableefficacy. On the other hand, the serum concentration exceeds the EC90for just over ½ the dosing interval when MMV665917 is dosed at 30 mg/kgtwice daily. This likely explains the variable efficacy of MMV665917when given at this dose.

FIG. 3 shows results for in vitro dose-response curves with MMV665917and the other MMV compounds tested in the NOD SCID gamma mouse model, asshown in FIG. 2. Results are combined data from two independentexperiments, expressed as average percent inhibition and SD.

Conclusions.

Based on its efficacy in the NOD SCID gamma immunocompromised mousemodel, MMV665917 is a promising lead compound for treatment ofcryptosporidiosis. The in vivo efficacy of MMV665917 is surprising andunexpected, given its in vitro potency. A number of compounds withgreater in vitro potency and high systemic exposure following oraldosing in mice have no activity in the NOD SCID gamma mouse model.

Example 2

This example describes the evaluation of compounds of the presentdisclosure towards parasites.

To assess if MMV665917 is cidal or static for C. parvum and determinethe concentration of compound required to maximize the rate of parasiteelimination, C. parvum persistence in HCT-8 colonic carcinoma cells wasassayed in the presence of increasing multiples of the in vitro EC90.The assay strategy and predictions are summarized in FIG. 6. Briefly,the assay infects HCT-8 intestinal epithelial cells with the C. parvumIowa strain as in Example 1. Parasites are then allowed to develop for24 hours prior to the addition of the indicated concentration ofexperimental compounds (time 0 in the graphs above). At the indicatedtimes following compound addition, samples are then fixed, the hostcells and parasites are fluorescently stained, and parasites areenumerated by automated epifluorescence microscopy and image process asdescribed in Example 1.

FIG. 7A shows the results for the control compound nitazoxanide, whichlacks activity in the NOD SCID gamma immunocompromised mouse model andin immunocompromised people. The compound appears to be static, since C.parvum persists indefinitely in the presence of as much as 12× the EC₉₀concentration. MMV665917 (FIG. 7B), on the other hand, appears to becidal for Cryptosporidium, since parasites are eliminated atconcentrations equal to or exceeding the EC₉₀. The maximal rate ofparasite elimination is achieved at a compound concentration of 3×EC₉₀.Furthermore, since additional increases in the compound concentrationhave no effect on the rate of parasite elimination, the mode-of-actionof MMV665917 appears to be time-dependent, rather than concentrationdependent.

Example 3

This example details pharmacokinetic (PK) studies of a compound of thepresent disclosure.

The pharmacokinetics of MMV665917 were studied in overnight-fasted maleSprague Dawley rats that had access to water ad libitum throughout thepre- and post-dose sampling period, and access to food was re-instated 4h (hour) post-dose.

MMV665917 was administered intravenously as a 10 min (minute) constantrate infusion via an indwelling jugular vein cannula (1 mL per rat, n=2rats) and orally by gavage (10 mL/kg per rat, n=2 rats).

Samples of arterial blood and total urine were collected up to 24 hpost-dose. Arterial blood was collected directly into borosilicate vials(at 4° C.) containing heparin, Complete® (a protease inhibitorcocktail), potassium fluoride, and EDTA to minimise potential for exvivo degradation of test compound in blood/plasma samples. Oncecollected, blood samples were centrifuged, supernatant plasma wasremoved and stored frozen (−20° C.) until analysis by LC-MS.

Formulation Preparation and Analysis.

Formulations were prepared on the day of dosing and animals were dosedwithin 45 minutes of preparation.

IV Formulation. MMV665917 was dissolved in propylene glycol and ethanolbefore Milli-Q water was added producing a clear solution. Theformulation was filtered through a 0.22 μm syringe filter prior todosing, and the measured concentration of compound the filtered solutionwas 0.24 mg/mL.

Oral Formulation. MMV665917 was wet milled in HPMC-SV in an agate mortarand sonicated for 10 minutes producing a uniform suspension. The bulkformulation was mixed by inverting the tubes prior to drawing eachdosing volume and the average measured concentration of compound inaliquots (n=3) of formulation was 0.32 mg/mL (range 0.28-0.37 mg/mL).

In Vitro Determination of Plasma/Blood Stability and WholeBlood-to-Plasma Ratio. MMV665917 was spiked into fresh heparinized wholeblood (collected from male Sprague Dawley rats) or plasma of acorresponding volume (which acted as a matrix control) to an initialnominal concentration of 500 ng/mL. As a whole blood assay for thecompound was not available, the concentration of MMV665917 in the spikedplasma (i.e. matrix control) was used as a surrogate measurement of thespiked whole blood concentration. Whole blood samples were incubated at37° C. for up to 240 min after which plasma and erythrocytes wereseparated by centrifugation prior to determination of plasmaconcentrations by LC-MS.

Whole blood-to-plasma partitioning ratios (B/P) were obtained bydividing the measured concentration in the plasma control sample by theconcentration measured in plasma following centrifugation of wholeblood.

Results and Discussion.

MMV665917 was stable in freshly collected rat plasma and whole bloodwhen incubated in vitro at 37° C. (Table 2 in FIG. 4) suggesting thatblood-mediated degradation not likely to contribute significantly to thein vivo clearance of the compound. The apparent in vitro whole blood toplasma partitioning ratio (B/P) of MMV665917 was 1.1.

No adverse reactions or compound related side effects were observedfollowing IV or oral administration of MMV665917. Evidence of haemolysiswas observed in plasma following IV administration, which can beattributed to the high organic content of the formulation vehicle (40%(v/v) propylene glycol and 10% (v/v) ethanol) which was required forsolubilisation.

Plasma concentration versus time profiles of MMV665917 following IV andoral administration are presented in FIG. 1 and plasma concentrationsfrom each rat are provided in Appendix 3. Pharmacokinetic parametersdetermined via non-compartmental analysis are summarized in Table 1 inFIG. 4.

Following IV administration, plasma concentrations remained above theanalytical LLQ for 16 h and the apparent terminal half-life wasapproximately 3-4 h. The apparent volume of distribution was moderateand in vivo clearance was low. The fraction of the dose recovered inurine as intact MMV665917 over the 24 h sampling period wasapproximately 25%, suggesting that direct urinary excretion is asignificant in vivo elimination route for this compound in rats.

Following oral administration, MMV665917 was slowly absorbed withmaximum plasma concentrations being observed at 5 h post-dose and theapparent terminal half-life was consistent with that observed after IVdosing. The apparent oral bioavailability ranged from 24% to 43%. On thebasis of the low in vivo blood clearance, it is likely that MMV665917would be subject to low hepatic first pass elimination suggesting thatoral exposure may be limited by solubility and/or permeability.Furthermore, given the consistency in the IV profiles, the approximatelytwo-fold difference in oral exposure between rats may be indicative ofvariability in absorption-related processes. Further studies in agreater number of animals would be required to better define the basisfor the observed variability.

FIG. 4 shows physical characteristics and mouse pharmacologic data forMMV665917. The compound shows moderate systemic exposure with slowintestinal absorption (i.e., the time to peak serum concentrationfollowing a single dose is 6 hours) and a prolonged serum half-life.

PK data for MMV665917 was also tested orally in rats. The compoundexhibited an apparent terminal half-life of approximately 3-4 h, amoderate volume of distribution and low in vivo clearance. Followingoral administration, the compound was slowly absorbed and apparentbioavailability was approximately 30%.

MMV665917 is absorbed from the intestine relatively slowly andincompletely, resulting in both prolonged intestinal exposure andsystemic exposure. The unusual PK characteristics of MMV665917 may be asignificant factor enabling in vivo efficacy.

Example 4

This example details additional pharmacokinetic studies of plasmaconcentration of MMV665917 on mice, rats and calves afteradministration.

The concentration of MMV665917 in mouse, rat and dairy calf plasmafollowing a single dose is shown in FIG. 8. Compounds were dosed asindicated either orally by oral gavage or intravenously, and serumsamples were collected at the indicated time points. The MMV665917plasma concentration was measured by LC-MS/MS. The dashed line shows thepredicted plasma level out to 24 hours for mice, which was determinedusing the calculated elimination half-life.

The actual and simulated mouse and calf plasma concentrations forMMV665917, correlated to the EC90 and 3×EC90 concentrations are shown inFIG. 9. Mouse serum concentrations measured from a single 55 mg/kg oraldose of MMV665917 (FIG. 9) were used to simulate the mouse serumconcentrations following BID dosing of either 30 mg/kg or 60 mg/kg. Themeasured calf plasma concentration following a single 22 mg/kg oral doseis also shown. Note that a minimum concentration of EC90 was requiredfor parasite elimination in vitro, and the rate of parasite eliminationin vitro was maximized at 3× the EC90 concentration. Following 30 mg/kgBID dosing in mice, this concentration is not attained and MMV665917 wasvariably efficacious. A 60 mg/kg BID dose, on the other hand, results ina sustained plasma concentration greater than the EC90, and is curativein the NOD SCID gamma mouse model.

Although the present disclosure has been described with respect to oneor more particular embodiments and examples, it will be understood thatother embodiments and examples of the present disclosure may be madewithout departing from the scope of the present disclosure.

What is claimed is:
 1. A method for treating an individual diagnosedwith or suspected of having a Cryptosporidium infection or prophylaxisin an individual who is at risk of having a Cryptosporidium infectioncomprising administering to the individual a composition comprising atherapeutically effective or a prophylactically effective amount of acompound having the following structure:

wherein R⁴ is selected from the group consisting of hydrogen atom, C₁-C₉alkyl group, and haloalkyl groups; R⁵ is selected from the groupconsisting of substituted or unsubstituted benzyl group, substituted orunsubstituted phenyl group, substituted or unsubstituted phenoxy group,and substituted or unsubstituted aniline group.
 2. The method of claim1, wherein R⁵ is selected from the group consisting of


3. The method of claim 1, wherein the compound is selected from thegroup consisting of:


4. The method of claim 1, wherein the cryptosporidium infection iscaused by Cryptosporidium parvum, Cryptosporidium hominis, orCryptosporidium andersoni.
 5. The method of claim 1, wherein theindividual is human or a non-human animal.